CYP3A5-mediated metabolism of midazolam in recombinant systems is highly sensitive to NADPH-cytochrome P450 reductase activity

Abstract

1. Data from in vitro drug metabolism studies with recombinant enzyme systems are frequently used to predict human drug metabolism in vivo. However, for the CYP3A probe substrate midazolam (MDZ), considerable variability in enzyme kinetic parameters has been observed in different in vitro studies. The aim of this study was to explore the effect of varying activities of the electron donor NADPH-cytochrome P450 reductase (CPR) on CYP3A5-mediated metabolism of MDZ.

2. Microsomes with similar levels of CYP3A5 but 12-fold difference in CPR activity showed a 30-fold difference in intrinsic clearance for the formation of 1′-OH-MDZ. Significantly higher K_m and lower V_max for the formation of 1′-OH-MDZ were found in microsomes with low CPR activity compared with microsomes with higher CPR activity (P = 0.024 and 0.001). In the microsomes with lowest CPR activity, the formation of 1′-OH-MDZ displayed Michaelis–Menten kinetics, whereas substrate inhibition was observed in the two preparations with higher CPR activity.

3. The present study shows that the CPR activity in different recombinant enzyme preparations is crucial for in vitro CYP3A5-mediated clearance of MDZ. This suggests that the CPR activity of enzyme preparations could be an important factor for the ability of in vitro data to predict human drug metabolism in vivo.

Keywords::

• In vitro metabolism
• midazolam
• recombinant CYP3A5
• cytochrome P450 reductase

Acknowledgements

The authors acknowledge Prof. Eva Skovlund for help in the statistical analysis and Mrs. Siri Johannessen for her excellent technical assistance. Thanks to Hoffman La Roche for kindly giving us MDZ.

Declaration of interest

The authors report no conflicts of interest.